1. Field of the Invention
This invention relates to use of a method for screening nonpathogenic anti-inflammatory bacterial strains, and products and methods using such strains for treatment and prophylaxis of inflammation caused by gastrointestinal bacteria such as Helicobacter pylori, other species of Helicobacter, and other inflammation-causing gastrointestinal pathogens.
2. Description of the Related Art
Helicobacter pylori is a spiral-shaped bacterium that colonizes the stomach by, among other things, its ability to produce urease to neutralize the acids in the stomach. Urease converts urea, of which there is an abundant supply in the stomach, to bicarbonate and ammonia, which are strong bases. This results in a cloud of acid-neutralizing bases around the H. pylori cells, protecting them from the acid in the stomach. The H. pylori cells penetrate and traverse the gastric mucus layer and attach to epithelial cells in the lining of the stomach. At least some strains of H. pylori have the ability to produce toxins. Infection with H. pylori activates the host immune system, which sends white blood cells, killer T cells and other infection-fighting agents to the area, but the body's immune system is not effective in reversing the effects of H. pylori in the mucus lining of the stomach. The H. pylori cells remain in the lining, and the immune system escalates its response to the cells, creating an inflammation if there are not sufficient anti-inflammatory mechanisms available. During the infection with H. pylori, cytokine intercellular signal proteins generated by the host epithelium dendritic cells, natural killer cells, T-cells and other immune defense cells propagate the immune response to the invading pathogen. Consequently, host neutrophils are attracted to and infiltrate the stomach epithelium and persist there throughout the infection. These cells generate, among other factors, reactive oxygen products, such as superoxide radicals, which lead to oxidation in the epithelial cells and consequent epithelial cell death, ulcer formation and ultimately carcinogenesis. H. pylori also induces leakage of nutrients from the host over the stomach epithelium providing a nutrient source to sustain the H. pylori cells and exacerbate the infection and its consequences. H. pylori is able to evade the human immune system and survive in the stomach despite the immune response of the host and the mechanisms of this evasion are the subject of current research.
Current therapy is based on eradicating H. pylori through antibiotics and proton pump inhibitors rather than attempting to eliminate the effects of excessive immune response of the host to the infection, such as making sufficient anti-inflammatory mechanisms available, which is the purpose of the present invention.
Thus, infection with H. pylori causes an increased risk of developing gastritis, gastric and duodenal ulcers, including peptic ulcer, gastric cancer, and gastric mucosa-associated lymphoid tissue lymphoma. These problems are not caused directly by the H. pylori cells, but by the inflammation of the stomach lining in response to the H. pylori. Various treatments have been used to ameliorate the symptoms of gastric and duodenal ulcers, such as treatments that reduce acid production in the stomach, combined with antibiotics. Novel vaccines against H. pylori has also been tried but with limited success. It is also known that other species of Helicobacter, as well as other gastrointestinal pathogens, can cause gastrointestinal inflammation.
In a recent research article, researchers studying H. pylori infections concluded that infection by H. pylori elicits gastric mucosal sialylation as part of the chronic inflammatory response and that many virulent strains are thus better able to attach to the inflamed site (Science 18:573-578, 2002).
Inflammation in the stomach and gastrointestinal tract is mediated by intercellular signal proteins known as cytokines which are produced by macrophages and dendritic cells in the epithelium in response to an antigenic stimulus such as that produced by H. pylori or other pathogens. Upon contact between the epithelium and the antigen such as H. pylori or endotoxins produced by it, such as LPS, antigen presenting cells (including dendritic cells) in the epithelium propagate the signal to naive macrophages which then respond in a so-called Th-1 type response where pro-inflammatory cytokines including TNFα, IL-1, IL-6, IL-12 are produced by the macrophages. These cytokines in turn stimulate natural killer cells, T-cells and other cells to produce interferon γ (IFNγ), which is the key mediator of inflammation. IFNγ leads to an escalation of the inflammatory response and the reactions described above that lead to cytotoxicity. Naive macrophages can also respond to antigens with a Th-2 type response. This response is suppressed by IFNγ. These Th-2 type cells produce anti-inflammatory cytokines such as IL-4, IL-5, IL-9 and IL-10.
IL-10 is known to inhibit the production of IFNγ and thus dampen the immune response. The balance between Th-1 and Th-2 type cells and their respective cytokine production defines the extent of the inflammation response to a given antigen. Th-2 type cells can also stimulate the production of immunoglobulins via the immune system. Anti-inflammatory activity in the gastrointestinal tract, where there is a reduced TNFα level, correlates with enhanced epithelial cells (gut wall lining integrity) and thus to a reduction in the negative effects caused by gastrointestinal pathogens and toxins.
The results of a number of research studies indicate that DNA can exert an anti-inflammatory action on intestinal epithelial cells, or can stimulate the immune system. (Madsen et al. and Rachmilewitz et al, respectively, presentations at Digestive Disease Week, May 19-22, 2002, The Moscone Center, San Francisco).
Mice spontaneously develop chronic colitis, which does not occur in germ-free animals. Mouse colitis is similar to human Crohn's disease, a chronic serious inflammatory disease of the gastrointestinal tract. Crohn's disease usually occurs in the intestines, but may occur anywhere in the gastrointestinal tract. These conditions require the presence of enteric bacteria and are both Th1-mediated-IL-12-dependent forms of colitis. Because of the similarities of the causes and symptoms, mouse models of colitis and other mouse models are used to study components of the inflammatory response directly, and are, as the same mechanisms apply in man, accepted to be used to develop treatments for human gastrointestinal disease.
Lactobacillus reuteri is one of the naturally occurring inhabitants of the gastrointestinal tract of animals and is routinely found in the intestines of healthy animals and despite the low pH, occasionally also in the human stomach. It is known to have antibacterial activity. See, for example U.S. Pat. Nos. 5,439,678, 5,458,875, 5,534,253, 5,837,238, and 5,849,289. When L. reuteri cells are grown under anaerobic conditions in the presence of glycerol, they produce the antimicrobial substance known as reuterin (β-hydroxy-propionaldehyde).
L. coryniformis is a less well-known species of Lactobacillus which is a rather common inhabitant of the human oral cavity. It can also be found in soil, manure and plant material. It has been found in silage and as a beer spoiler, and good lactic acid production has been reported as well as antifungal activity. The L. coryniformis MM7 isolate (ATCC PTA-4660) used herein was found in human mother's milk.
Immunomodulating activity has also been associated with various lactobacilli. While the possibility of effective antibacterial activity by several lactobacilli is known, it was not previously known that substantial differences existed between strains in their ability to reduce gastrointestinal inflammation, nor that such strains could be selected.
It is therefore an object of the invention to provide strains of Lactobacillus which have been selected for their capability of reducing gastrointestinal inflammation, such as that due to Helicobacter pylori. It is a further object of the invention to provide products containing said strains, including agents for treatment or prophylaxis of inflammation associated with Helicobacter pylori for administration to humans, including conditioned media in which said strains have grown and protein-containing extracts thereof.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.